Abstract
Although the C-terminal domain (DIII) of three-domain Cry insecticidal toxins from Bacillus thuringiensis has been implicated in various biological functions, its exact role still remains to be elucidated. Here, the 21-kDa isolated DIII fragment of the 65-kDa Cry4Ba mosquito-specific toxin was analyzed for its binding characteristics toward lipid-bilayer membranes. When the highly-purified Cry4Ba-DIII protein was structurally verified by attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, it revealed the presence of a distinct β-sheet structure, corresponding to its structure embodied in the Cry4Ba crystal structure. Binding analysis via surface plasmon resonance (SPR) spectroscopy revealed that the 21-kDa Cry4Ba-DIII truncate displayed tight binding to immobilized liposome membranes in a two-step manner, exhibiting a dissociation rate constant (kd) comparable to the 65-kDa full-length toxin. Also similar to the Cry4Ba full-length toxin, its isolated DIII truncate was able to anchor a part of its molecule into the immobilized membrane as the SPR signal was still detected after prolonged treatment with proteinase K. However, unlike the full-length active toxin, the DIII truncate was unable to induce membrane permeability of calcein-loaded liposomes or ion-channel formation in planar lipid bilayers. Together, our present data have disclosed a pivotal role of C-terminal DIII in serving as a membrane anchor rather than a pore-forming moiety of the Cry4Ba mosquito-active toxin, highlighting its potential mechanistic contribution to the interaction of the full-length toxin with lipid membranes in mediating toxicity.
Highlights
Strains of Bacillus thuringiensis (Bt), a Gram-positive bacterium producing insecticidal proteins known as crystal (Cry) and/or cytolytic (Cyt) δ-endotoxins [1], have been widely used as highly specific and safe bio-insecticides for the control of agricultural pests and human disease vectors [2,3].Of particular interest, the Cry4Ba mosquito-active toxin produced from Bt subsp. israelensis is toxic to the larvae of Aedes and Anopheles spp., vectors of important tropical infectious diseases of dengue hemorrhagic fever and malaria, respectively [3,4,5]
The Cry4Ba mosquito-active toxin produced from Bt subsp. israelensis is toxic to the larvae of Aedes and Anopheles spp., vectors of important tropical infectious diseases of dengue hemorrhagic fever and malaria, respectively [3,4,5]
It should be noted that the 21-kDa Cry4Ba-DIII protein most likely exists as a monomer since its elution profile from the size-exclusion FPLC analysis revealed that the purified truncate was eluted as a single peak in line with the elution volume of the 23-kDa lysozyme protein marker (Figure 2A)
Summary
Strains of Bacillus thuringiensis (Bt), a Gram-positive bacterium producing insecticidal proteins known as crystal (Cry) and/or cytolytic (Cyt) δ-endotoxins [1], have been widely used as highly specific and safe bio-insecticides for the control of agricultural pests and human disease vectors [2,3]. The functional importance of both DI and DII of the two closely related mosquito-specific toxins (i.e., Cry4Aa and Cry4Ba) was intensively investigated [12,13,14,15,16,17,18]. The 21-kDa isolated DIII fragment from the Cry4Ba mosquito-active toxin was shown to be capable of binding along the apical microvilli of A. aegypti larval midgut, conceivably participating in toxin interactions with either lipid membranes or target protein receptors [28]. We have further demonstrated the functional significance of C-terminal DIII of Cry4Ba for serving as a tight-binding lipid anchor, highlighting its potential contribution to toxin interactions with the bilayer membrane to mediate toxicity
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